The Gcd10p/Gcd14p complex is the essential two-subunit tRNA(1-methyladenosine) methyltransferase of Saccharomyces cerevisiae

The modified nucleoside 1-methyladenosine (m(1)A) is found at position 58 i n the T Psi C loop of many eukaryotic tRNAs. The absence of m(1)A from ail tRNAs in Saccharomyces cerevisiae mutants lacking Gcd10p elicits severe def ects in processing and stability of initiator methionine tRNA (tRNA(i)(Met) ). Gcd10p is found in a complex with Gcd14p, which contains conserved motif s for binding S-adenosyl-methionine (AdoMet). These facts, plus our demonst ration that gcd14 Delta cells lacked m(1)A, strongly suggested that Gcd10p/ Gcd14p complex is the yeast tRNA(m(1)A)methyltransferase [(m(1)A)MTase]. Su pporting this prediction, affinity-purified Gcd10p/Gcd14p complexes used Ad oMet as a methyl donor to synthesize m(1)A in either total tRNA or purified tRNA(i)(Met) lacking only this modification. Kinetic analysis of the purif ied complex revealed KM values for AdoMet or tRNA(i)(Met) Of 5.0 mu M and 2 .5 nM, respectively. Mutations in the predicted AdoMet-binding domain destr oyed GCD14 function in vivo and (m(1)A)MTase activity in vitro. Purified Fl ag-tagged Gcd14p alone had no enzymatic activity and was severely impaired for tRNA-binding compared with the wild-type complex, suggesting that Gcd10 p is required for tight binding of the tRNA substrate. Our results provide a demonstration of a two-component tRNA MTase and suggest that binding of A doMet and tRNA substrates depends on different subunits of the complex.